The purpose of this study was to develop a mobile learning acceptance model for pre-service teachers and to examine the relationships among technology acceptance factors. The literature on mobile learning acceptance lacks studies on pre-service teachers and studies that include concrete mobile learning scenarios. To overcome these problems, we have developed and implemented a mobile-technology-enabled information technology course. The data collection and analysis were conducted in two separate studies. First, we developed a mobile learning acceptance scale and applied confirmatory factor analysis with 408 participants. The final instrument included 28 items measuring eight technology acceptance factors, namely behavioral intention, attitude towards use, perceived usefulness, perceived ease of use, social influence, facilitating conditions, self-efficacy, and anxiety. After this, we collected a new set of data from 316 participants to examine the relationships among the factors using structural equation modeling. In both studies, we investigated the respective models’ invariance across gender and discipline groups, and both models fulfilled invariance requirements. The results indicated that perceived ease of use and social influence have direct effects on behavioral intention, whereas self-efficacy has an indirect effect. Depending on the group, the explained variance of behavioral intention ranged between 18.1% and 60.6%.